1. Field of the Invention
The present invention relates to organic compounds which may be used as ligands for the preparation of water-soluble complexes of lanthanides or of certain transition metals, to a process for preparing the same, and to the use thereof as fluorescent probes.
2. Description of Related Art
Lanthanide ion complexes have very particular spectroscopic properties that allow applications in the field of the detection by luminescence. These complexes have a very wide Stokes shift, very fine emission lines, of the order of a few nm, which are characteristic of the lanthanide ion used. They can emit in the visible or near infrared region, and have an extremely long lifetime of the excited state, which may be up to a millisecond. This last feature is an essential asset: it allows time-resolved detection (which makes it possible to eliminate the parasite fluorescence signals) and brings about a very large increase in the detection sensitivity of the complexes by luminescence microscopy or in fluoroimmunological analyses. Luminescent lanthanide ion complexes consequently have applications in the majority of the field of conventional fluorescence.
However, lanthanide ion complexes are generally difficult to obtain. Many properties of the complex are dependent on the structure of the ligand and of the lanthanide ion, especially the excitation efficacy of the complex, the degree of stability of the lanthanide complexation in competitive chemical medium and in serum medium (which must be high to prevent release of the cations), the quantum luminescence yield and the possibility of forming covalent bonds with the material to be labeled for the biological applications of the complexes. Adequate excitation of the complexes may be obtained when the ligand of the complex comprises heteroaromatic groups whose function is to capture light and transfer it to the lanthanide ion which will reemit. This phenomenon is known as the antenna effect. The choice of these heteroaromatic groups defines many spectroscopic properties of the final complex, especially the spectral excitation range and the quantum luminescence yield.
CN-1811429-A describes a complex of Tb3+ and of a ligand which has a 2,6-dipyrazolylpyridine backbone in which each of the pyrazolyl groups bears a —CH2—N(CH2CO2H)2 group. Said complex is useful for detecting singlet oxygen. It is obtained via a process which consists in attaching the anthracene group to a dibromo-aminopyridine, and then in modifying the pyridyl group by reacting the bromine atoms with suitable reagents to replace each Br with a pyrazolyl group bearing a —CH2—N(CH2CO2H)2 group.
EP-0 770 610 describes lanthanide ion complexes in which the ligand is a 2,6-dipyrazolylpyridine backbone in which each of the pyrazolyl groups bears a —CH2—N(CH2CO2H)2 group. The preparation process consists in first preparing a dibromo 2,6-dipyrazolylpyridine compound, which is then modified to obtain the two —CH2—N(CHG2CO2H)2 end groups. This process does not make it possible to obtain compounds in which the pyridyl group bears substituents chosen to adjust the properties of the lanthanide complex in which said compounds constitute the ligand.
FR-2 935 973 describes ligands derived from 2,6-dipyrazolylpyridine in which each of the pyrazolyl groups bears a —CH2—N(CHG2CO2R)2 group in which each of the R groups represents H or an alkali metal or a quaternary ammonium group, the pyrazolyl groups also possibly bearing one or two substituents chosen from an alkyl group containing from 1 to 4 carbon atoms, or alternatively these two substituents together form a diradical forming an aromatic ring with the two carbon atoms that bear them. These ligands are capable of complexing lanthanide ions and find applications in labeling and biphotonic microscopy. However, they have poor solubility in water and great instability in purely aqueous medium, in saline medium or in purely biological medium.